Enhancing structural diversity of terpenoids by multisubstrate terpene synthases

• Min Li and
• Hui Tao

Beilstein J. Org. Chem. 2024, 20, 959–972, doi:10.3762/bjoc.20.86

• generation of 29, a variant of FgMS-D510A with an inactive PT domain, rather than wild-type FgMS, was used in combinatorial biosynthesis. Furthermore, critical residues controlling substrate specificity were identified using site-directed mutagenesis. Interestingly, when the aromatic residue Phe65 was

Substrate specificity of a ketosynthase domain involved in bacillaene biosynthesis

• Zhiyong Yin and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2024, 20, 734–740, doi:10.3762/bjoc.20.67

• [28][29] was exchanged through site-directed mutagenesis, resulting in the BaeJ-KS2-C222A enzyme variant. After heterologous expression and protein purification (Figure S1, Supporting Information File 1), the same protocol for the incubation with (S)-11 and (R)-11 followed by exchange of the

Elucidating the glycan-binding specificity and structure of Cucumis melo agglutinin, a new R-type lectin

• Jon Lundstrøm,
• Emilie Gillon,
• Valérie Chazalet,
• Nicole Kerekes,
• Antonio Di Maio,
• Ten Feizi,
• Yan Liu,
• Annabelle Varrot and
• Daniel Bojar

Beilstein J. Org. Chem. 2024, 20, 306–320, doi:10.3762/bjoc.20.31

• protein) with optimized codons for Escherichia coli was synthesized flanked by NcoI and XhoI restriction sites where L6 was mutated to valine. The gene was inserted in the homemade plasmid pET40b-TEV where the enterokinase cleaving site was replaced by a TEV cleavage site by site directed mutagenesis

Functions of enzyme domains in 2-methylisoborneol biosynthesis and enzymatic synthesis of non-natural analogs

• Binbin Gu,
• Lin-Fu Liang and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2023, 19, 1452–1459, doi:10.3762/bjoc.19.104

• diphosphate synthase (FPPS) and 2MIBS from Streptomyces coelicolor [26] (Scheme 1B). Crystal structures of both enzymes have been obtained [27][28] and allowed for a deep structure-based investigation of 2MIBS through site-directed mutagenesis [29]. The predicted amino acid sequences of 2MIBS homologs from

Germacrene B – a central intermediate in sesquiterpene biosynthesis

• Houchao Xu and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2023, 19, 186–203, doi:10.3762/bjoc.19.18

• pristinaespiralis 1 is an intermediate in the cyclisation of farnesyl diphosphate (FPP) to selina-4(15)-7(11)-diene [36]. Several SdS enzyme variants have been constructed by site-directed mutagenesis, including the enzyme variants D83E, E159D and W304L, for which the product spectrum is shifted towards 1 as the

Characterization of a new fusicoccane-type diterpene synthase and an associated P450 enzyme

• Jia-Hua Huang,
• Jian-Ming Lv,
• Liang-Yan Xiao,
• Qian Xu,
• Fu-Long Lin,
• Gao-Qian Wang,
• Guo-Dong Chen,
• Sheng-Ying Qin,
• Dan Hu and
• Hao Gao

Beilstein J. Org. Chem. 2022, 18, 1396–1402, doi:10.3762/bjoc.18.144

• Talaromyces wortmannii ATCC 26942. Heterologous expression reveals that TadA catalyzes the formation of a new fusicoccane-type diterpene talaro-7,13-diene. D2O isotope labeling combined with site-directed mutagenesis indicates that TadA might employ a different C2,6 cyclization strategy from the known

• . Keywords: cytochrome P450 enzyme; diterpene synthase; gene cluster; genome mining; site-directed mutagenesis; Introduction Terpenoids are a large class of natural products that attract extensive attention, due to not only their potential applications in pharmaceuticals, agrochemicals, etc. but also due to

Enzymes in biosynthesis

• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2022, 18, 1131–1132, doi:10.3762/bjoc.18.116

• as Escherichia coli or Saccharomyces cerevisiae. Besides in vitro studies with purified enzymes, heterologous expressions of whole pathways for the production of compounds is possible [6]. Enzyme mechanisms can be addressed through structure-based site-directed mutagenesis, which may also lead to

Targeting active site residues and structural anchoring positions in terpene synthases

• Anwei Hou and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2021, 17, 2441–2449, doi:10.3762/bjoc.17.161

• are otherwise highly conserved. Site-directed mutagenesis experiments for these residues are reported that showed different effects, resulting in some cases in an improved catalytic activity, but in other cases in a loss of enzyme function. For other enzyme variants a functional switch was observed

• mechanisms; isotopes; site-directed mutagenesis; terpenes; Introduction Terpenoids now span more than 90,000 known compounds, which makes them by far the largest class of natural products [1]. Despite this fact, all compounds are made from only two C5 building blocks, dimethylallyl diphosphate (DMAPP) and

• residue (pyrophosphate sensor, located usually 46 residues upstream of the NSE triad, Figure 1) can be observed. Site-directed mutagenesis demonstrated that this residue is important for SdS catalysis [11]. Additional conserved residues include a Pro at the bottom of helix C causing a helix turn (21

Current understanding and biotechnological application of the bacterial diterpene synthase CotB2

• Ronja Driller,
• Daniel Garbe,
• Norbert Mehlmer,
• Monika Fuchs,
• Keren Raz,
• Dan Thomas Major,
• Thomas Brück and
• Bernhard Loll

Beilstein J. Org. Chem. 2019, 15, 2355–2368, doi:10.3762/bjoc.15.228

• site-directed mutagenesis studies [36][38], which showed that, whereas D110 and D111 are crucial for catalysis, the solvent exposed D113 is not. The here described structural situation is different compared to TPSs with a canonical DDXXD motif. For instance in the structure of epi-isozizaene synthase

• binding of GGDP and the cyclization reaction. Based on 2H- as well as 13C-isotope labeling experiments a surprising reaction mechanism has been derived (Scheme 2) [35]. Strong support for the proposed reaction mechanism has been predominantly provided by site-directed mutagenesis of amino acids with an

• Figure 7). F107 has been targeted by site-directed mutagenesis as well (Table 2 and Scheme 1) [30], leading to compounds R-cembrene A (7) and cyclooctat-1,7-diene (8). Now the cationic intermediate has two possibilities to react to G, either by a 1,3- and 1,5-hydride shift or by two 1,2-hydride shifts

Targeting the Pseudomonas quinolone signal quorum sensing system for the discovery of novel anti-infective pathoblockers

• Christian Schütz and
• Martin Empting

Beilstein J. Org. Chem. 2018, 14, 2627–2645, doi:10.3762/bjoc.14.241

• site-directed mutagenesis combined with thermodynamic profiling, as well as surface plasmon resonance (SPR) experiments with and without covalent active site blockade, corroborated that the nitrophenylmethanol class directly binds to the active site near the reactive cysteine of PqsD [55]. This is in

Enzymatic synthesis of glycosides: from natural O- and N-glycosides to rare C- and S-glycosides

• Jihen Ati,
• Pierre Lafite and
• Richard Daniellou

Beilstein J. Org. Chem. 2017, 13, 1857–1865, doi:10.3762/bjoc.13.180

• substrates can also be envisioned. In parallel, our knowledge of the enzymatic mechanisms has allowed us to modify and improve the original activities through reasoned site-directed mutagenesis. However, despite major advances, all the rules that finely tune the biocatalysts are still poorly understood and

• directed mutagenesis of the potent residues [64]. In these cases, the mutated enzymes are no longer able to perform the hydrolysis of the substrates. The use of external and suitable nucleophilic anions such as azide, formate or acetate allows the rescue of the activity and can also represent an efficient

• , such enzymatic approaches can nowadays efficiently be utilized in particular for the preparation of pure and well-defined complex glycoproteins [63]. Use of external nucleophiles The identification of the two amino acid side chains in both retaining and inverting GHs is usually performed through site

Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems

• Katarina Kemper,
• Max Hirte,
• Markus Reinbold,
• Monika Fuchs and
• Thomas Brück

Beilstein J. Org. Chem. 2017, 13, 845–854, doi:10.3762/bjoc.13.85

• of highly functionalized compounds. Novel approaches discussed in this review include metabolic engineering as well as site-directed mutagenesis to expand the natural terpene landscape. Focusing mainly on the validation of successful integration of engineered biosynthetic pathways into optimized

• , combinatorial enzyme design and microbial engineering. Mutational engineering of terpene synthases Site-directed mutagenesis of diterpene cyclases is conventionally applied to elucidate structure–function relationships and mostly targets the active site of the enzyme in order to change the polarity or dimension

• -directed mutagenesis (indicated through wrench) of TPS (green) for product modulation or introduction of a linker-coding sequence for co-expression of P450 monooxygenase and reductase (blue and red); 4: Heterologous expression in E. coli (depicted in orange). Construction of synthetic operons and screening

Polyketide stereocontrol: a study in chemical biology

• Kira J. Weissman

Beilstein J. Org. Chem. 2017, 13, 348–371, doi:10.3762/bjoc.13.39

• -oxopentanoate to an ACP, has not been tested)). In any case, these data encouraged the view that mutation of a few key residues in the KR active sites might be used to alter reduction stereochemistry. Site-directed mutagenesis can indeed modify the stereochemical outcome of ketoreduction, at least in vitro

• based on the crystal structure of the DEBS DH [92], in which the conserved His acts as a general base to deprotonate at C-2, while an Asp residue serves as a general acid to stabilize the C-3 hydroxy leaving group. However, only the His has been shown by site-directed mutagenesis to be essential [111

• is most often valine, but also alanine or phenyalanine. The role of these residues in stereocontrol was evaluated in vivo by site-directed mutagenesis of a derivative of DEBS 1-TE in which the KR domain of module 2 was replaced with the ‘reductive loops’ (DH-KR-ER tridomains) sourced from the DEBS

Posttranslational isoprenylation of tryptophan in bacteria

• Masahiro Okada,
• Tomotoshi Sugita and
• Ikuro Abe

Beilstein J. Org. Chem. 2017, 13, 338–346, doi:10.3762/bjoc.13.37

• , the region is thus no longer aspartate-rich, and so hereafter it is referred to as a pseudo-SARM. A site-directed mutagenesis analysis of the ComQRO-E-2 from strain RO-E-2 with an in vitro geranylation reaction revealed that the lone-conserved second aspartate residue in the pseudo-SARM of ComQ is

Computational methods in drug discovery

• Sumudu P. Leelananda and
• Steffen Lindert

Beilstein J. Org. Chem. 2016, 12, 2694–2718, doi:10.3762/bjoc.12.267

• are associated with diseases, contributing to increase or decrease of target activity. Binding sites in target proteins can be experimentally determined; for example using site-directed mutagenesis or X-ray crystallography. There are also a variety of computational binding pocket identifying

Artificial Diels–Alderase based on the transmembrane protein FhuA

• Hassan Osseili,
• Daniel F. Sauer,
• Klaus Beckerle,
• Marcus Arlt,
• Tomoki Himiyama,
• Tino Polen,
• Akira Onoda,
• Ulrich Schwaneberg,
• Takashi Hayashi and
• Jun Okuda

Beilstein J. Org. Chem. 2016, 12, 1314–1321, doi:10.3762/bjoc.12.124

• –Alderases reported so far used soluble proteins, where the binding site of Cu(II) was formed either by site-directed mutagenesis [22][23], by incorporation of a suitable ligand, or copper complex in an apo-protein [24][25][26][27]. Here we report on the use of the robust transmembrane protein Ferric

Cyclisation mechanisms in the biosynthesis of ribosomally synthesised and post-translationally modified peptides

• Andrew W. Truman

Beilstein J. Org. Chem. 2016, 12, 1250–1268, doi:10.3762/bjoc.12.120

• (possibly 21, as RiPPs containing selenocysteine were proposed in a recent bioinformatic study [18]). Excitingly, the ribosomal origin of RiPPs means that significant chemical changes to complex natural products can be achieved by simple site-directed mutagenesis. This requires the associated tailoring

• sensitive to AS-48 [140]. A gene cluster has been identified [141], and site-directed mutagenesis has been used to identify key residues in the precursor peptide that are critical for cyclisation [142], but the actual cyclase has not been characterised. One explanation for the limited understanding of this

Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions

• Paul P. Kelly,
• Anja Eichler,
• Susanne Herter,
• David C. Kranz,
• Nicholas J. Turner and
• Sabine L. Flitsch

Beilstein J. Org. Chem. 2015, 11, 1713–1720, doi:10.3762/bjoc.11.186

• interactions [23], whereas Met184 is part of the P450cam substrate recognition site 2 (SRS 2) [24]. Accordingly, the entire P450cam active site was partitioned into seven residue pairs which were targeted in site-directed mutagenesis experiments in the manner of CASTing (Figure 1) [25]. NDT codon degeneracy

A bivalent glycopeptide to target two putative carbohydrate binding sites on FimH

• Thisbe K. Lindhorst,
• Kathrin Bruegge,
• Andreas Fuchs and
• Oliver Sperling

Beilstein J. Org. Chem. 2010, 6, 801–809, doi:10.3762/bjoc.6.90

• trisaccharide substructures, mainly α-D-Man-(1→3)-[α-D-Man-(1→6)]-D-Man. By employing site directed mutagenesis, it was found that mutations in one of these cavities significantly reduces binding, indicating that this could be a second carbohydrate binding site, relevant for ligand binding [21]. Thus, it was